Studies were initiated to define the genetic basis of attenuation of the parainfluenza virus type 3 (PIV3)cp45 live attenuated vaccine candidate. The PIV3 cp45 candidate vaccine (cold-passage 45) is safe, immunogenic, and phenotypically stable in seronegative human infants and currently is in Phase II trials in young vaccinees. The determination of the genetic basis of attenuation of cp45 was a complex task since the candidate virus has 15 mutations that encode three phenotypes, namely the attenuation (att), temperature-sensitive (ts), and cold-adaptation (ca) phenotypes. A reverse genetics system was used successfully to construct a recombinant cp45 (rcp45) virus that contained all fifteen cp45-specific mutations, and this recombinant was essentially indistinguishable from the biologically-derived cp45. A series of recombinant viruses were constructed that contained a single cp45 mutation or combination of mutations allowing us to scan the viral genome and assign the ts, ca, and/or att phenotype to specific mutations. The determination of the genetic basis of attenuation of cp45 was a major accomplishment because it provided sound genetic evidence to explain the high level of attenuation and stability of cp45 previously observed in animals and humans. These findings provided a firm scientific basis for continuing to use the cp45 mutant as a basis for vaccines against the other PIVs (PIV1 and PIV2) known to be associated with respiratory tract disease in humans. A live attenuated PIV1 vaccine for parainfluenza virus type 1 (PIV1), a major cause of croup in infants and young children, was generated by replacement of the HN and F protective antigens of the PIV3 cp45 vaccine candidate with their PIV1 counterparts using the techniques of reverse genetics. The rPIV3-1 cp45 vaccine candidate is attenuated and immunogenic in hamsters and clinical lots are being prepared. A modified version of the PIV3 cp45 vaccine was produced in case cp45 does not prove to be sufficiently attenuated in ongoing trials in humans. A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live attenuated respiratory syncytial virus (RSV) candidate vaccine, specifies the ts and attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly-related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of cp45 was mutagenized to leucine (F456L) to yield rcp45-456. rcp45-456 was 5-fold more restricted in the respiratory tract of chimpanzees than rcp45 and induced, like cp45, a moderate to high level of PIV3-specific serum antibodies. Thus, we have identified a mutation that can be imported to further attenuate cp45 if ongoing clinical trials indicate that this is needed. - parainfluenza virus, croup, pneumonia, bronchiolitis, vaccines, live-attenuated virus vaccines, childhood immunization